Plantago asiatica mosaic virus: An emerging plant virus causing necrosis in lilies and a new model RNA virus for molecular research

Abstract Taxonomy Plantago asiatica mosaic virus belongs to the genus Potexvirus in the family Alphaflexiviridae of the order Tymovirales. Virion and genome properties Plantago asiatica mosaic virus (PlAMV) has flexuous virions of approximately 490–530 nm in length and 10–15 nm in width. The genome of PlAMV consists of a single‐stranded, positive‐sense RNA of approximately 6.13 kb. It contains five open reading frames (ORFs 1–5), encoding a putative viral polymerase (RdRp), movement proteins (triple gene block proteins, TGBp1‐3), and coat protein (CP), respectively. Host range PlAMV has an exceptionally wide host range and has been isolated from various wild plants, including Plantago asiatica, Nandina domestica, Rehmannia glutinosa, and other weed plants. Experimentally PlAMV can infect many plant species including Nicotiana benthamiana and Arabidopsis thaliana. It also infects ornamental lilies and frequently causes severe necrotic symptoms. However, host range varies depending on isolates, which show significant biological diversity within the species. Genome diversity PlAMV can be separated into five clades based on phylogenetic analyses; nucleotide identities are significantly low between isolates in the different clades. Transmission PlAMV is not reported to be transmitted by biological vectors. Virions of PlAMV are quite stable and it can be transmitted efficiently by mechanical contact. Disease symptoms PlAMV causes red‐rusted systemic necrosis in ornamental lilies, but it shows much weaker, if any, symptoms in wild plants such as P. asiatica. Control Control of the disease caused by PlAMV is based mainly on rapid diagnosis and elimination of the infected bulbs or plants.


| INTRODUC TI ON
Plantago asiatica mosaic virus (PlAMV), genus Potexvirus, is an emerging virus originally described in 1976 from the weedy plant Plantago asiatica in the Russian Far East
For almost three decades no other natural hosts were known until infections of edible lilies were reported in Japan and subsequently in commercially produced ornamental lilies in the Netherlands. Since then, PlAMV infection has become widespread through the commercial lily trade, and additional natural hosts have been reported from various countries.
Over the last two decades there has been significant work on molecular and biological characterization of PlAMV. Here we summarize current knowledge regarding PlAMV, including its natural and experimental host range, strain differentiation, host interactions, and utility as a plant viral vector to examine virus-host interactions.

| Natural host range
PlAMV was first reported from P. asiatica, a perennial herbaceous species endemic to north-eastern Asia, by . P. asiatica readily establishes in disturbed soils and can be a weed in fields and gardens. For the next 25 or more years, P. asiatica was the only known natural host. However, a potexvirus was reported to infect cultivated plants of the ornamental shrub N. domestica (heavenly bamboo) in California, USA (Moreno et al., 1976), and later was named Nandina mosaic virus (Zettler et al., 1980). Finally, it was classified as an isolate of PlAMV when its complete genome sequence was determined (Hughes et al., 2005).
Additional natural hosts began to emerge in the early 2000s. (Lilium leichtlinii var. maximowiczii) in Japan (Komatsu et al., 2008;Ozeki et al., 2006;Sasaki, 2008) and Primula sieboldii (Komatsu et al., 2008) were followed later by the emergence of PlAMV in the commercial lily trade. This was reported first in the Netherlands (EPPO, 2011) and in Chile in 2013 (Vidal et al., 2016).
(MZ344590) in Canada. The natural hosts are shown in bold in Table 1.

| Experimental host range
When PlAMV was first discovered in P. asiatica, a partial experimental host range was determined, including 24 species from 12 plant families ; Table 1). However, it remained unknown whether other isolates have a similar host range or not. Recently, the experimental host range of a lily isolate, one of the "European-type" PlAMV isolates in commercial lilies, was determined (Hammond & Rane, 2022), which identified an additional 20 species representing 12 taxonomically diverse plant families not previously reported Zettler et al., 1980; see Table 1). Another recent study showed that PlAMV isolates in distinct phylogenetic clades show differential infectivity to several experimental hosts. Among five isolates tested, only two isolates each can systemically infect Arabidopsis thaliana or P. asiatica (authors' unpublished data). Several experimental hosts listed in Table 1 as either locally or systemically susceptible to a lily PlAMV isolate were reported as not susceptible to a nandina isolate (Zettler et al., 1980). Collectively, these findings suggest that PlAMV has a wide host range but that different isolates vary in their ability to infect some hosts.

| Transmission
No biotic vector of PlAMV is known. As with other potexviruses, PlAMV is readily transmitted by mechanical inoculation with sap extracts (Conijn, 2014;De Kock, 2013), but also spreads rapidly between infected and previously healthy lilies planted in a common container by uptake and probably exudation through the roots, and is remarkably stable in contaminated planting media (Conijn, 2014;De Kock, 2013).
PlAMV is also transmitted between lilies during bulb washing and packing, which may be the major route of infection in commercial lilies (Chastagner et al., 2017;De Kock, Kok, et al., 2013;De Kock, Slootweg, et al., 2013). Fields in which PlAMV-infected lilies were previously grown can retain viable virus, able to infect up to 8% of lily stocks previously thought to be free from PlAMV (De Kock, Slootweg, et al., 2013). PlAMV can also systemically infect lily plants by mechanical inoculation or sap injection into stems (Tanaka et al., 2019).

| Symptoms
The natural hosts of PlAMV vary in the degree of symptom expression.
Naturally infected V. grypoceras showed obvious mosaic symptoms (Komatsu et al., 2017). N. domestica from Japan showed primarily leaf narrowing ( Figure 1c; Komatsu et al., 2017), while that found in the United States showed the systemic mosaic without leaf distortion on the first or second leaves produced after inoculation, and intermittently on subsequently developing nandina leaves (Zettler et al., 1980). New Zealand spinach + +  Note: Host range is from experimental inoculation by Hammond and Rane (2022) (Tzanetakis et al., 2005;Yamaji et al., 2001). The nucleotide identity of the whole genome between PlAMV and TVX is almost 70%, below the demarcation criteria for distinct potexvirus species (72%), but it is one of the highest identities between different species of the genus (Komatsu et al., 2008;Yamaji et al., 2001). TVX has recently been detected from ornamental lily cultivars from which PlAMV has been repeatedly detected (Jo & Cho, 2018). Although there are no reports of intermediate virus isolates of these closely related species, PlAMV and TVX may be considered as a phylogenetically related group of monocotinfecting potexviruses.

| Genome diversity of PlAMV isolates
Phylogenetic analysis showed that PlAMV isolates affecting ornamental lilies worldwide ("European" isolates) are highly genetically homogenous, suggesting a common origin of these isolates (Hammond & Reinsel, 2018). However, PlAMV isolates, in general, have genomic diversity within the species. As stated above, PlAMV has been isolated from a variety of weed plants, including P. asiatica  Solovyev et al., 1994), N. domestica (Hughes et al., 2005;Komatsu et al., 2017), and R. glutinosa (Kwak et al., 2018;Uehara-Ichiki et al., 2018). These PlAMV isolates from plants other than ornamental lilies share less than 85% nucleotide identities with lilyinfecting European isolates (Hammond & Reinsel, 2018;Komatsu et al., 2017), therefore the ancestral host plant from which lilyinfecting isolates were derived is still unclear. Our phylogenetic analysis based on the full-length genomic sequences of PlAMV isolates showed that they were divided into five distinct clades according to their geographical origins and host plants (authors' unpublished data). Nucleotide identities between PlAMV isolates belonging to different clades are less than 85%. Sequence variability is dispersed throughout the genome, while several insertions/ deletions of amino acids were concentrated within the linker region between methyltransferase and helicase domains of the replicase (Komatsu et al., 2017). Recent study has also revealed several positively selected amino acid residues in PlAMV-encoded proteins, including this linker region (authors' unpublished data). Further studies are needed to identify specific amino acids contributing to intraspecies diversification and adaptation to ornamental lilies, and to understand the evolutionary history of PlAMV leading to genetic diversification within the species. Indeed, a PlAMV "replicon", which consists of only the replicase ORF flanked by 5′-and 3′-UTRs, can produce minus-strand genomic RNA (Komatsu et al., 2011). This indicates that both UTRs have essential cis-elements required for interaction with the replicase, as is the case with PVX (Komarova et al., 2006;Kwon et al., 2005;Kwon & Kim, 2006;Park et al., 2013).

| Genome organization and gene expression
Mechanisms of gene expression of these five ORFs are basically similar to those reported in PVX (Verchot, 2021). Replicase is translated directly from genomic RNA , while other proteins are translated from three subgenomic RNAs (sgRNAs). However, recent study revealed that only two sgR-NAs are detected from PlAMV-infected plants, sgRNA1 (about 1.9 kb in length) and sgRNA2 (about 0.8 kb), and movement proteins TGBp1-TGBp3 are mainly translated from a single sgRNA1, which encodes TGBp1 as the 5′-terminal ORF, by leaky scanning (Fujimoto et al., 2022;Figure 2a). This leaky scanning is promoted through a short 5′-UTR of sgRNA1 and the Kozak sequence around its initiation codon (Fujimoto et al., 2022). Similar to other potexviruses, CP encoded in the most 3′-terminal ORF5 is likely to be translated from sgRNA2.  . On infection with PlAMV-GFP, GFP is expressed from sgRNA2 as a fusion protein (Figure 2b).
An sgRNA duplication strategy, used for the development of other potexvirus vectors (Abrahamian et al., 2021), is difficult to apply to PlAMV due to its overlapping ORF4 and ORF5 .

| Systemic necrosis
In general, defence responses against plant viruses consist of those mediated by NLR (nucleotide-binding and leucine-rich repeat) proteins and RNA silencing (Moon & Park, 2016 (Seo et al., 2006).

Inoculation of chimeric viruses between Li1 and Li6 showed that
the systemic necrosis was determined by cysteine at amino acid residue 1154 of Li1 replicase (Ozeki et al., 2006). However, agroinfiltration studies revealed that the elicitor activity of PlAMV replicase resides in its helicase domain (HEL), not its RNA-dependent RNA polymerase domain (POL) that contains the amino acid residue 1154 (Komatsu et al., 2011). Notably, the necrosis-eliciting activity of HEL was also observed in Li6, and inducible-expression analysis demon-

| RNA silencing
Plant viruses encode VSRs that inhibit various steps of host antiviral RNA silencing (Csorba et al., 2015). The first identified VSR of potexviruses was TGBp1 of PVX, which interferes with spread of the RNA silencing signal (Voinnet et al., 2000). TGBp1s of several potexviruses were shown to possess varying degrees of VSR activity, among which that of PlAMV isolate Li1 was relatively strong   (Brosseau & Moffett, 2015;Jaubert et al., 2011).
Another study showed that susceptibility of A. thaliana to PVX varies depending on the natural variation of AGO2 (Brosseau et al., 2020).
These findings suggest that TGBp1 of PlAMV, which can effectively infect A. thaliana, also inhibits activities of these DCLs and AGOs in addition to the RDR6-SGS3 complex.
In addition to a dcl2/dcl4Arabidopsis mutant that is more susceptible to multiple plant viruses, an ago4 mutant was more susceptible to PlAMV infection, which suggests that DCL2/DCL4 and AGO4 restrict PlAMV infection (Brosseau et al., 2016). Functional analyses using transient expression assays demonstrated that cytosolic AGO4 is involved in this restriction (Brosseau et al., 2016). Experiments on the additional target(s) of PlAMV TGBp1, and comparison of VSR activity between PlAMV and PVX are needed to reveal the role of TGBp1 in successful viral infection.

| RE S IS TAN CE G ENE S EFFEC TIVE AG AIN S T Pl AMV
No cultivars of ornamental lilies, in which PlAMV causes severe economic losses, have yet been identified that are completely PlAMVresistant, although symptom expression and viral infectivity depend on the cultivars (Tanaka et al., 2019). In contrast, laboratory experiments that use PlAMV-based GFP-expression vector and the model plant species A. thaliana have identified several host genes that confer resistance against, or effectively suppress, PlAMV infection . These include dominant and recessive resistance genes as well as other defence-related genes.

| Dominant resistance genes
Jacalin-type lectin required for potexvirus resistance 1 (JAX1) is a dominant resistance factor that restricts PlAMV at the single-cell level (Yamaji et al., 2012). JAX1 is a noncanonical lectin-type resistance protein, not a conventional NLR. An active JAX1 was found from five of gene RTM1 of A. thaliana, which confers resistance against tobacco etch virus, but the spectrum of resistance of JAX1 and RTM1 was different; JAX1 confers resistance against potexviruses in general, while RTM1 is effective against potyviruses (Chisholm et al., 2001;Yamaji et al., 2012). An in vitro replication assay based on evacuolated BY-2 protoplast extracts revealed that JAX1, but not RTM1, restricts replication of potexviruses by targeting the massive protein complexes required for viral replication . This targeting is mediated via interaction with the viral replicase, and a single amino acid substitution, Q336H, allows PVX infection in JAX1-expressing plants (Sugawara et al., 2013). However, the same mutation in PlAMV severely decreases infectivity in plants either with or without JAX1, suggesting that JAX1-mediated resistance does not easily produce resistance-breaking viral variants (authors' unpublished data). Jacalinrelated lectin genes are widely found in plants and many are involved in disease resistance (Esch & Schaffrath, 2017). However, it remains to be determined whether the antiviral functions of jacalin-related lectins, including JAX1 and RTM1, are conserved in other plants.

| Recessive resistance genes
In addition to the dominant resistance genes, genetic screening of ethyl methyl sulfonate-mutagenized Arabidopsis lines with PlAMV-GFP revealed recessive resistance genes that encode a plant factor required for successful virus infection (Hashimoto, Neriya, Yamaji, et al., 2016). EXA1 (essential for potexvirus accumulation 1) is the first identified recessive resistance gene against PlAMV infection and inhibits replication at the single-cell level (Hashimoto, Neriya, Keima, et al., 2016). EXA1 contains a GYF domain and an eIF4Ebinding motif. As the translation initiation factor eIF4E is the bestknown recessive resistance gene against plant viruses, EXA1 may form a translation initiation complex with eIF4E and possibly exerts its function through regulation of translation of PlAMV replicase or of a host factor required for PlAMV replication (Hashimoto, Neriya, Keima, et al., 2016). EXA1 orthologs are found in a wide range of plant species, including tomato, rice, and N. benthamiana, and knockdown of EXA1 orthologs in tomato and N. benthamiana significantly reduced the accumulation of potexviruses and the related lolavirus.

This restriction of viral infection is cancelled by complementation
with the rice EXA1 gene, indicating that the proviral function of EXA1 is conserved among a wide range of plants (Yusa et al., 2019).
However, the effect of EXA1 knockdown in N. benthamiana on virus accumulation differs depending on virus species, suggesting that EXA1 paralogs function redundantly in a virus-specific manner. Interestingly, EXA1 (also referred to as PSIG1) was reported to restrict PCD during bacterial and oomycete infections (Matsui et al., 2017). Localization of PSIG1 (EXA1) to P-bodies supports its role in the suppression of P-body activity, such as translational arrest of viral genomic RNA or nonsense-mediated decay (Mäkinen et al., 2017).

Another recessive resistance gene against PlAMV found from
A. thaliana is nCBP1, an isoform of eIF4E, known to be the loss-ofsusceptibility gene for multiple plant viruses (Hashimoto, Neriya, Yamaji, et al., 2016). nCBP1 is required for infection of plant viruses in the families Alpha-and Betaflexiviridae. Whereas nCBP1 is not required for replication at the single-cell level, it is required for cell-tocell movement of PlAMV (Keima et al., 2017). Accumulation of both TGBp2 and TGBp3 was decreased in the ncbp mutant, which causes the inhibition of cell-to-cell movement (Keima et al., 2017).

| Other defence-related genes
Similar to various other RNA viruses, potexviruses require intracellular membranes for replication. Confocal laser scanning microscopy has revealed that the replicase of PVX is associated with ER membranes (Tilsner et al., 2013). Similarly, membrane association is important for replication of PlAMV (Komatsu et al., 2021;Yoshida et al., 2019). Membrane-associated replication can cause elevated membrane stress. Indeed, ER-localized TGBp3 of PVX was shown to induce unfolded protein responses (UPR), enhancing proteinfolding capacity at ER, especially of the IRE1/bZIP60 pathway (Gaguancela et al., 2016). As well as the IRE1/bZIP60 pathway, the IRE1-independent bZIP17 pathway functions to restrict early stages of PlAMV infection in Arabidopsis plants, indicating that the two arms of UPR signalling inhibit the accumulation of PlAMV (Gayral et al., 2020). Meanwhile, bZIP60 and bZIP28 induce genes that support PlAMV infection, suggesting that plants have intricate regulatory mechanisms of UPR on virus infection (Herath et al., 2020).
PlAMV-specific polyclonal antisera have been prepared against the purified virus of either lily or nandina isolates (see Hammond, 2018) or against the bacterially expressed CP of a lily isolate (Chen et al., 2013). These antisera have been used for immunodiffusion tests (Zettler et al., 1980), direct tissue blotting, and indirect, antigen-coated plate enzyme-linked immunosorbent assay (ELISA; Chen et al., 2013), double-antibody sandwich ELISA (DAS-ELISA; e.g. Hammond et al., 2015;Parrella et al., 2015), or rapid lateral flow assays (LFAs). Some commercial agricultural diagnostic companies produce ELISA reagent kits and/or LFAs for PlAMV detection.
For greatest sensitivity in DAS-ELISA testing of lilies, testing leaves at the time of flowering, using leaves from about three-quarters of the height of the flowering stem is recommended, although it can also be used on roots and bulb-scales of stored bulbs preplanting.
Notably, both ELISA and LFAs have been found to detect a wide variety of PlAMV isolates from different phylogenetic clades.
Multiple groups have reported reverse transcriptionpolymerase chain reaction (RT-PCR) detection of PlAMV, using either generic potexvirus primers (van der Vlugt & Berendsen, 2002) followed by sequencing or various PlAMV-specific primers, mainly derived from the replicase-or the CP-encoding regions (e.g., Chen et al., 2013;Hammond et al., 2015). RT-PCR can detect PlAMV in some samples not detected by DAS-ELISA (Hammond et al., 2015). Kim et al. (2019) incorporated a pair of PlAMV-specific primers with primer sets specific for CMV, LMoV, and LSV to detect these four lily-infecting viruses. Multiplex RT-PCR assays have been developed to detect PlAMV, CMV, LMoV, and LSV in lilies (Xu et al., 2021), and PlAMV and four other viruses in R. glutinosa (Kwon et al., 2019). An immunocapture (IC)-RT-PCR assay, applied to detect three lily-infecting viruses, CMV, LMoV, and LSV, is also promising , but IC-RT-PCR has not been reported for PlAMV detection.
Real-time quantitative RT-PCR (RT-qPCR) has also been used and is more suitable for quantifying PlAMV titre than other assays. Furthermore, a multiplex TaqMan RT-qPCR system for simultaneous detection of PlAMV, CMV, LSV, LMoV, and shallot yellow stripe virus in lilies has recently been reported (Xu et al., 2021). In this case, primers and probes were designed from conserved regions of the CP genes of each virus, and the probes for each virus were labelled with a different fluorescent dye. The sensitivity of the multiplex reaction was equal to that of each uniplex assay and can be applied for the comprehensive detection of viruses from lily production fields (Xu et al., 2021).
A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed (Komatsu et al., 2015) and shown to detect diverse isolates of PlAMV with a 10-fold increase in sensitivity over conventional RT-PCR, without requiring RNA purification. Pricking the leaf sample with a toothpick, followed by dipping it into the reaction mix, resulted in reliable detection in field samples (Komatsu et al., 2015).
One of the most cost-effective assays for simultaneous detection of PlAMV and other lily-infecting viruses is a macroarray prepared on a nylon filter membrane with probes for each virus (PlAMV, CMV, LMoV, and LSV; Sugiyama et al., 2008), which showed similar or greater sensitivity than ELISA and correctly identified mixed infections.
High-throughput sequencing has been used to identify PlAMV and any associated viruses infecting P. asiatica (Lim et al., 2016), lilies (e.g., Jo & Cho, 2018;Xu et al., 2017), and R. glutinosa (Uehara-Ichiki et al., 2018), yielding several nearly complete genomes.  Slootweg, et al., 2013). To minimize this possibility the bulb lots of highest quality should be treated before bulb lots known to have a higher prevalence of PlAMV infection; the processing equipment itself should also be decontaminated and the wash water treated to minimize transmission in washing subsequent lots (Chastagner et al., 2017;Conijn, 2014;De Kock, 2013;De Kock, Slootweg, et al., 2013). Frequent decontamination of the tools and equipment used at other stages of production is also recommended.

| Control strategies
As PlAMV is highly stable and can also be retained in the soil or plant parts, planting in soil or growing medium in which infected plants have previously been grown should be avoided (Chastagner et al., 2017;De Kock, Slootweg, et al., 2013). Heating of contaminated planting medium for a sufficient time at a high temperature will inactivate PlAMV, with a temperature of 65°C maintained for 10 min recommended for bulb wash water (Conijn, 2014), if that is practical.
Weed control in fields where PlAMV-infected plants are, or have been, grown is also important as a number of weed species have been found to maintain infectivity, as have volunteer plants regrowing after harvest of the crop (Chastagner et al., 2017;De Kock, Slootweg, et al., 2013). Maintaining fields fallow for one planting season may minimize sources of infection for the next crop (De Kock, Slootweg, et al., 2013). Moreover, the milled sphagnum used to pack lily bulbs for shipping has been proven to carry PlAMV (authors' unpublished data) and should be disposed of with caution to avoid contamination.
The possibility of using plant defence activators to minimize PlAMV infections has been studied by Matsuo et al. (2019) using ASM, a functional analog of salicylic acid, which can inhibit infection of tobacco mosaic virus (Chivasa et al., 1997;Murphy & Carr, 2002).
Treating N. benthamiana with ASM prior to inoculation with PlAMV reduced the number of infection foci compared to controls, reflecting inhibition of replication, but did not affect cell-to-cell movement; however, there was a delay in long-distance movement into the uninoculated leaves (Matsuo et al., 2019). Future work to further understand the mechanisms may lead to more effective prevention or minimization of the effects of plant virus infection.

| CON CLUS ION
The economic losses suffered in the ornamental lily industry and the rapid spread of PlAMV through the international trade in lily bulbs spurred interest in research into this rapidly emerging virus.
To date, however, the natural host of origin of the strain in commercial lilies remains unidentified but seems to be derived from a single introduction.
The extent of the work on PlAMV that has resulted from this interest has revealed several features that make PlAMV an attractive

ACK N OWLED G EM ENTS
We are grateful to Dr T. Arie, Takashi Moriyama, and Tsutomu Moriyama for their kind support and discussion. We thank Dr T. Uehara-Ichiki for pictures of R. glutinosa. We thank the Special

Research Fund of the Institute of Global Innovation Research at
Tokyo University of Agriculture and Technology (GIR-TUAT), Japan.
This work was supported in part by Grants-in-Aid for Scientific Research 19 K06048 for K.K. from the Japan Society for the Promotion of Science.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing are not applicable to this article as no new data were created or analysed.